Experiments have been initiated to study the chemical mechanism of the autoprocessing of the human immunodeficiency virus (HIV-1) protease (PR) from fusion proteins. An assay has been defined in which the wild-type fusion protein undergoes time-dependent autoprocessing concomitant with the appearance of HIV-1 PR activity. Analysis of the time course of the reaction monitored by immunoblotting showed the initial appearance of an intermediate product of cleavage of the fusion protein. This reaction was determined to be first-order from initial rate measurements of the appearance of enzymatic activity, and the disappearance of the full-length fusion protein. Pepstatin A inhibits autoprocessing with an inhibition constant that is at least five-fold lower than the inhibition constant for the mature PR. These results are consistent with a mechanism in which the processing event to release HIV-1 PR activity from the fusion protein involves intramolecular cleavage at the amino terminus from a dimer polyprotein. Inhibition of this reaction by pepstatin A and HIV-1 PR substrate indicates that the substrate binding site is intact in the full- length polyprotein. The other approach involves structure to function characterization of HIV-1 polyprotein processing by mutational analysis. By characterizing the in vitro processed products of the purified full-length HIV-1 PR fusion proteins which contain mutations at either the N- or the C-terminal cleavage sites, we have identified mutations which completely block one of the cleavage sites of the PR and effect cleavage at the non-mutated wild- type site. In addition, we show that the sequence specificity for cleavage is more stringent at the N-terminus of the PR than at the C-terminus. A rapid method has been described by which the PR can be expressed and purified to yield enzyme with high specific activity. A buffer system has been established for the stabilization of the PR that would allow kinetic and dimerization studies in solution. Production of the enzyme has aided in two detailed collaborative studies: (i) Studies on the effect of salt on the kinetic parameters of retroviral proteases in comparison with mammalian aspartic acid proteases; and (ii) Kinetic studies of the subsite specificity of HIV-1 and HIV-2 proteinases.